JP2002520298A - Targeted site-specific drug delivery compositions and methods of use - Google Patents

Abstract

(57) Summary The present invention relates to pharmaceutical compositions and methods for delivery of therapeutic agents. The methods and compositions of the present invention can achieve site-specific delivery of therapeutic agents, especially for agents such as oligonucleotides that have presented problems in reaching targeted sites at the required therapeutic levels. Allows for lower doses and improved efficacy. The targeted delivery of ultrasound can be used to enhance the release of the therapeutic agent. The delivery system encapsulated gas-filled microbubbles with protein formed in an environment containing no N ₂ depletion or N ₂ and the like. These microbubbles are smaller and more stable than microbubbles sonicated in the presence of air.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates to pharmaceutical compositions and methods for delivering bioactive agents. The methods and compositions of the present invention can be used to achieve site-specific delivery of biologically active substances. This specificity allows for lower drug doses and improved efficacy,
Particularly for drugs such as oligonucleotides, which typically suffer from the problem of achieving therapeutic concentrations in the targeted organ, allowing for lower drug doses and improved efficacy.

BACKGROUND OF THE INVENTION Drug delivery technology is used in drug therapy to increase drug availability, to reduce drug doses, and to reduce drug-induced side effects. . These techniques serve to control, regulate, and target the release of drugs in the body. Current drug delivery goals are to achieve less frequent drug administration, maintain constant and sustained therapeutic levels of the drug in the systemic circulation or at specific target organ sites, reduce undesirable side effects, and achieve desired therapeutic benefits And reduced dose concentrations required for Ultimately, a method that non-invasively targets delivery of a desired drug to a target organ is desired.

[0003] To date, drug delivery systems include proteins, polysaccharides, synthetic polymers, erythrocytes,
Drug carriers based on DNA and liposomes were mentioned. New generations of biologics (eg, monoclonal antibodies, gene therapy vectors, anticancer agents (eg, taxol), virus-based drugs, and oligonucleotides (ODNs) and polynucleotides) present several problems with delivery. I have. Indeed, drug delivery can be a major obstacle to achieving mainstream therapeutic use of these drugs, and the initial potential of these drugs appeared to be unlimited, but their therapeutic parameters Hampered the realization of full profits.

[0004] The use of synthetic oligodeoxyribonucleotides (ODNs) that have been chemically modified to confer nuclease resistance represents a fundamentally different approach to drug therapy. The most common application to date uses antisense ODNs that have a sequence that is complementary to a particular targeted mRNA sequence. The approach of antisense oligonucleotides to therapy involves the concept of simple and specific drug design, in which ODN causes mechanical intervention in the course of translation or earlier processing events. The advantage of this approach is its ability for gene-specific effects, which should be reflected in relatively low doses and minimal non-targeted side effects. However, phosphorothioate oligonucleotides, currently the most commonly used analogs in biological research, frequently display non-specific binding to proteins. In general, the major potential benefits of antisense approaches (low dose and minimal side effects) have been disappointing.

[0005] Drug delivery of oligonucleotides and polynucleotides has focused on two important challenges: transfection of oligonucleotides into cells and altered distribution of oligonucleotides in vivo. For transfection, biological approaches to improve cell uptake in vitro include:
It involved the use of vehicles such as liposomes and viral vectors (eg, reconstituted virus and pseudovirions). Methods for improving biodistribution have focused on drugs such as cationic lipids. This cationic lipid is
It is postulated that cell uptake of the drug is increased due to the attraction of positively charged lipids to the negatively charged surface of most cells.

[0006] Cationic liposomes have been reported to enhance gene transfer into vascular cells in vivo when administered by catheter (Muller, D.).
. W. Et al., Circ. Res. 75 (6): 1039-49, 1994). Cationic lipid DNA complexes have also been reported to result in efficient gene transfer into the lungs of mice after tracheal administration. Asialoglycoprotein poly (L) -lysine conjugates have experienced limited success as well as complexation with liposomes containing Sendai virus coat protein. Toxicity and biodistribution remain important issues.

From the foregoing, it is understood that more effective targeted drug delivery systems for the delivery of biologics, especially polynucleotides and oligonucleotides, are required for these drugs to achieve their full potential. Can be done.

SUMMARY OF THE INVENTION In one aspect, the invention provides a method for delivering a biological agent to a specific tissue site. The method comprises the steps of (a) forming an aqueous suspension of a plurality of microbubbles encapsulated with a protein and containing an insoluble gas, wherein the biological agent is in air. under conditions to produce N ₂ partial pressure in the small Awanai lower than N ₂ partial pressure of the resulting micro Awanai via sonication, is conjugated with the protein,; and (b This suspension is administered to an animal such that the protein directs the microbubble binding agent to the site of protein bioprocessing and, upon dissipation of the microbubble, Releasing the compound. Preferably, the microbubbles are N ₂ depletion environment, and more preferably is formed in an environment containing no N ₂ (e.g., oxygen).

In a preferred embodiment, the protein encapsulating the microbubbles is selected from the group consisting of albumin, human gamma globulin, human apotransferrin, β-lactose and urease, with albumin being particularly preferred. The insoluble gas is preferably a perfluorocarbon (eg, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, or perfluoropentane), with perfluoropropane and perfluorobutane being particularly preferred. The tissue site can be, for example, the liver or kidney of an animal.

[0010] Preferred biological agents conjugated to proteins include polynucleotides, oligonucleotides, nucleotides, and ribozymes, and naproxen, piroxicam, warfarin, furosemide, phenylbutazone, valproic acid, sulfisoxazole , Ceftriaxone, and miconazole. In selected embodiments, the biological agent is a polynucleotide, eg, an antisense oligonucleotide, an antigene oligonucleotide, an oligonucleotide probe, a vector, a viral vector or a plasmid.
A preferred class of polynucleotides includes phosphorothioate oligonucleotides.

The present invention also provides a related method for delivering a biological agent to a particular tissue site, the method comprising: (a) an aqueous suspension of microbubbles encapsulated with a protein and containing an insoluble gas; Forming a suspension, wherein the biological agent is conjugated to the protein; (b) administering the suspension to an animal; and (c) the tissue Exposing the site to ultrasound. Preferably, microbubbles are formed under conditions which produce N ₂ partial pressure in the small Awanai lower than N ₂ partial pressure of the micro Awanai obtained via sonication in air. Therefore, this microbubble is
Preferably formed in the N ₂ depleted environment, and more preferably free of N ₂ environment, for example, it is formed in oxygen.

In the practice of any of these methods, the suspension of microbubbles is preferably formed by performing the following steps: (a) 5% to 50% by weight, preferably about 5% by weight % Dextrose aqueous solution containing about 2% by weight dextrose.
Diluting an aqueous albumin solution containing from about 10% by weight, preferably about 5% by weight, of human serum albumin to about 2 times to about 8 times, preferably about 3 times; (b) stirring the solution with an insoluble gas. And (c) preferably in an N ₂ -depleted environment,
Exposing the solution to a sonication horn to create cavities at the microparticle sites in the solution, thereby producing stable microbubbles about 0.1-10 microns in diameter. Preferably, the N ₂ depleted environment of step (b) comprises:
Free environment N ₂ (e.g., oxygen), which is sonicated horn (son
It is blown into the interface between the icing horn and this solution.

[0013] In another aspect, the invention provides a microbubble composition useful for ultrasound imaging,
The composition comprises an aqueous suspension of a plurality of microbubbles containing encapsulated insoluble gas in proteins, wherein the micro the N ₂ partial pressure of the minute in the foam, which is sonicated in air less than N ₂ partial pressure of the foam. In a related aspect, the invention provides compositions useful for delivering a biological agent to a target site. The composition comprises an aqueous suspension of microbubbles having a biological agent conjugated to the protein.
Preferably, the microbubbles are no N _2. The preferred size of this microbubble is
It is 0.1-10 microns in diameter.

In these compositions, the protein is preferably selected from albumin, human gamma globulin, human apotransferrin, β-lactose and urease, with albumin being particularly preferred. The insoluble gas is preferably a perfluorocarbon (eg, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, and perfluoropentane), with perfluorobutane and perfluoropropane being particularly preferred. Biological agents suitable for conjugation to proteins that encapsulate microbubbles include oligonucleotides,
Polynucleotides and ribozymes, and naproxen, piroxicam,
Warfarin, furosemide, phenylbutazone, valproic acid, sulfisoxazole, ceftriaxone, and miconazole. Oligonucleotides are particularly preferred.

The present invention also provides a method of preventing carotid artery stenosis, particularly in animals that have suffered vascular trauma. The method comprises the steps of (a) forming an aqueous suspension of microbubbles containing an insoluble gas encapsulated in albumin, wherein the gas filling the microbubbles is N ₂ depleted or, preferably without N _2, and the albumin, the nucleic acid biological agent which inhibits the expression of a gene encoding a regulatory enzyme which mediates smooth muscle proliferation have been conjugated, step and, ( b) administering the suspension to an animal. A preferred method further comprises (c) exposing the trauma site to ultrasound to enhance the release of the biological agent. The inhibited gene is preferably c-myc or c-myb.

As described above, these compositions and methods can be used to significantly reduce the effective dosage of a biological agent, increase therapeutic indices, and improve bioavailability. This reduction in dose can also reduce the cytotoxicity and side effects of the drug. In addition, the present invention relates to other plasma binding drugs such as heparin, diltiazem, lidocaine, propanolol, cyclosporin,
And chemotherapeutic agents that require blood pool activation). For example, the anticoagulant properties of heparin can be dramatically improved by first combining the drug with dextrose / albumin, which has been exposed to orosomucoid-labeled perfluorocarbon and sonicated, and then administering the combination intravenously. It is shown herein that this can be enhanced.

[0017] The conjugate is designed for parenteral administration as an aqueous suspension. After administration and timed to coincide with the arrival of the injected bolus at the site of interest, energy can be administered in the form of acoustic waves, resulting in the creation of microbubble cavities; Released and delivered to the organ or other site of interest. The conjugation of albumin-encapsulated microbubbles or other protein-encapsulated microbubbles with biologics is accomplished by binding the biologic to specific tissues, including those that traditionally interact with proteins. It may allow for targeted delivery.

[0018] Improved gas-filled microbubbles are achieved by forming the microbubbles in the presence of a free environment N ₂ depleted environment or N _2. Such an environment produces microbubbles that are smaller, but more stable in venous and arterial blood. These smaller microbubbles result in greater diagnostic ultrasound contrast in the myocardium and, in therapeutic embodiments, may carry drugs to these areas with greater efficacy.

The present invention uses agents and methods traditionally used in diagnostic ultrasound imaging, and also provides a means for visualization of a therapeutic agent at its target site for delivery of the therapeutic agent. I do.

These and other objects and features of the present invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION I. Ultrasound Therapeutic Applications Ultrasound imaging has long been used as a diagnostic tool to assist in treatment procedures. This is based on the principle that acoustic energy can be focused on the area of interest and is reflected to produce an image. Generally, an ultrasound transducer is placed on the surface of the body overlying the area to be imaged, and the ultrasound energy generated by generating and receiving sound waves is transmitted. The ultrasound energy is reflected back to the transducer, where the ultrasound energy is translated into an ultrasound image. The amount and characteristics of the reflected energy depend on the acoustic properties of the tissue. Preferably,
Contrast agents that are echogenic are used to generate ultrasound energy in the area of interest and to improve received imaging. For a discussion of ultrasound-enhanced cardiography instruments, see DeJong, "Acoustic Properties of.
Ultrasound Contrast Agents ", Cip-Gege
vens Koninklijke Bibliotheek, Denhag (
1993), page 120 (see below).

Ultrasound-enhanced cardiography has been used to depict intracardiac structures, assess valve integrity, and demonstrate intracardiac anastomosis. Myocardial ultrasonography (MCE) has been used to measure coronary blood flow reserve in humans. MCE has been found to be a safe and useful technique for assessing relative changes in myocardial perfusion and delineating areas at risk.

Ultrasonic vibration has also been used at the therapeutic level in the medical field to increase the absorption of various drugs. For example, Japanese Patent Publication No. 52-1155591 discloses that transdermal absorption of a drug is enhanced by ultrasonic vibration. U.S. Patent Nos. 4,953,545 and 5,007,438 also disclose techniques for transdermal absorption of drugs with the aid of ultrasonic vibration. US Patent 5,315,9
No. 98 discloses a booster for drug treatment that includes microbubbles in combination with ultrasonic energy to allow diffusion and penetration of the drug. This reference is
In contrast to certain embodiments of the present invention, the use of therapeutic levels of ultrasound for up to 20 minutes is disclosed. Certain embodiments of the present invention enhance the release of conjugated bioactive agents using diagnostic levels of ultrasound, which are exposed for much less time.

In accordance with the present invention, a therapeutic agent is bound to a specifically designated site of interest using a traditional diagnostic ultrasound therapy contrast agent, and then the therapeutic agent is specifically targeted to the designated purpose. At the site of the drug, thereby altering the distribution of the drug. This objective can be achieved with the contrast agent alone and without the use of any diagnostic ultrasound. Such contrast agents are used in combination with traditional diagnostic ultrasound therapy (eg, including a peak negative pressure in the range of 0.1-3.5 MPa, and a transmission frequency in the range of 1.0-40 MHz). It is also demonstrated herein that it can enhance the release of the therapeutic agent at the site of interest. In a preferred embodiment of the method, low frequency ultrasound, i.e., a frequency below 1 MHz, most preferably from about 10 kHz to about 40 kHz
Are used.

II. Therapeutic Compositions The pharmaceutical compositions of the present invention comprise a liquid suspension of microbubbles, preferably 0.1 to 10 microns in diameter, comprising a blood insoluble gas, preferably an aqueous suspension. Contains turbid matter. The microbubbles are formed by encapsulating such gas microbubbles in a liquid.
The microbubbles may include various blood insoluble gases, such as fluorocarbon or sulfur hexafluoride gas. Generally, any insoluble gas that is non-toxic and gaseous at body temperature can be used. The gas also produces stable microbubbles having an average size diameter between about 0.1 and 10 microns when the pharmaceutical composition is sonicated to form microbubbles. Must be formed. Microbubbles having an average diameter in this range are suitable for transpulmonary passage and are sufficiently stable to prevent significant diffusion of gas within the microbubbles after intravenous injection and during transit to the target site. is there.

Insoluble gases must also have a lower diffusion coefficient and blood solubility than nitrogen or oxygen, which will diffuse at the internal pressure of the blood vessel. Examples of useful gases are carbon fluoride gas and sulfur hexafluoride. Generally, perfluorocarbon gases (eg, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, and perfluoropentane) are preferred. These gases, perfluorinated propane and perfluorinated butane, are particularly preferred because of their demonstrated safety for intraocular injection in humans (eg, Wong and Thompson, Ophthalmolog).
y 95: 609-613).

The gaseous microbubbles are stabilized by a filmogenic protein coating. Suitable proteins include naturally occurring proteins such as albumin, human gamma globulin, human apotransferrin, β-lactose and urease. The present invention preferably uses naturally occurring proteins, but synthetic proteins may also be used. Particularly preferred is human serum albumin. This protein is easily metabolized in the body,
And it is widely used as a contrast agent.

Preferably, an aqueous solution comprising a mixture of pharmaceutically acceptable sugars (eg, dextrose) is used in combination with the proteins described above. Examples of other saccharides suitable for use in the present invention are monosaccharides (those having the formula C ₆ H ₁₂ O ₆ (eg, hexose sugar, dextrose, fructose, or mixtures thereof)), disaccharides (formula C ₁₂ Those having H ₂₂ O ₁₁ (eg, sucrose, lactose, or maltose, or mixtures thereof), or polysaccharides (eg, of the formula (C ₆ H ₁₀
O ₅₎ soluble starch (here with _n, n is the whole number of between 20 and about 200) (e.g., amylase or dextran or mixtures thereof)
). However, saccharides are not required to achieve the results of the present invention.

The therapeutic agents of the invention are formulated in pharmaceutically effective dosage forms for peripheral administration to a host, optionally in combination with ultrasound therapy. Generally, such hosts are human hosts, but other mammalian hosts, such as dogs or horses, can also be subjected to this treatment.

III. Preparation of Microbubble Composition In a preferred embodiment, the composition of the present invention comprises commercially available albumin (human),
U. S. P. It is formulated from a mixture of a solution (generally supplied as a 5% or 20% by weight sterile aqueous solution) and commercially available dextrose (USP for intravenous administration). In a most preferred embodiment, the mixture comprises a two- to eight-fold dilution of a 2% to 10% solution of human serum albumin with a 5% to 50% solution of dextrose by weight.

[0031] Microbubbles are typically formed by sonication at room temperature and atmospheric pressure using an sonication horn. Such sonication causes cavitation in the dextrose / albumin solution at the site of particulate matter or gas in the fluid. These cavitation sites eventually resonate and produce small, undisrupted and stable microbubbles. During sonication, the solution is perfused with an insoluble gas, preferably a carbon fluoride gas, for about 80 seconds. Generally, about 4 × 10 ⁸
Microbubbles of a concentration greater than about 0.1-10 μm and preferably 5-6
Sonication conditions that produce a diameter of μm are preferred. The resulting microbubbles are concentrated at room temperature for at least about 120 ± 5 minutes, where the excess solution is placed in a sonicating syringe.

In an alternative method of preparation, as described in Example 2, 15 ± 2 ml of sonicated dextrose / albumin was manually added with 8 ± 2 ml of carbon fluoride gas prior to sonication. Stirred. The sonication is then allowed to proceed for 80 ± 5 seconds.

A third method of preparation (designed to improve the affinity of microbubbles for many plasma-binding drugs) is that of 5-30 mg of orosomucoid (α
-Acid glycoprotein) to the albumin / dextrose solution.

[0034] In accordance with the present invention, microbubbles, preferably, N ₂ depleted, preferably in an environment free of N _2, typically N ₂ depleted ultrasound or N ₂ without the gas (in comparison to air) It is formed by guiding to the interface between the processing horn and the solution.
As discussed further below, microbubbles formed in this manner are significantly smaller than microbubbles formed in air. These smaller microbubbles are more stable and
It results in improved delivery of therapeutic and diagnostic agents.

The microbubbles are then incubated with a biologically active agent, such that the drug is conjugated (typically by non-covalent attachment) to the protein-coated microbubbles. . Binding studies of oligonucleotides to albumin-coated microbubbles are described in Examples 4-7. These studies have shown, for example, that the binding is saturable, thus limiting non-specific interactions (
Example 6). In Example 7, the filmogenic proteins in the form of microbubbles (as conventionally used in contrast agents) retain their ability to bind compounds when the microbubbles are filled with an insoluble gas Is demonstrated. This finding is
In such microbubble contrast agents, this is contrary to previous reports that protein spheres are composed of denatured proteins. For example, US Pat. No. 4,572,203
No. 4,718,433, and U.S. Pat. No. 4,774,958. As demonstrated herein, an insoluble gas rather than air (
If (for example, perfluorocarbon) is used for the microbubbles, the protein retains its binding activity. This effect is probably due to the fact that most of the sonication energy is absorbed by the insoluble gas. Thus, a protein (eg, albumin) can bind to a biologically active drug to form a microbubble / drug conjugate. On the other hand, air-filled microbubbles probably do not retain their binding capacity much.

IV. Therapeutic Agents Therapeutic agents useful in the present invention are selected via their ability to bind to filmogenic proteins, and vice versa. For example, if the filmogenic protein is albumin, the therapeutic agent can include an oligonucleotide, a polynucleotide, or a ribozyme, all of which can bind to albumin and, as such, conjugate with microbubbles Can be formed. A list of drugs that bind to binding to albumin at site 1, which has been found to remain intact in the compositions and methods of the invention, is as follows:

[0037]

Embedded image If orosomucoid is added to dextrose / albumin, the list of drugs that can be potentiated also includes: erythromycin (antibiotic), lidocaine (antiarrhythmic), meperidine (analgesic), methadone (analgesic) ),
Verapamil and diltiazem (antianginal drugs), cyclosporine (immunosuppressant)
, Propanolol (antihypertensive and antianginal) and quinidine (antiarrhythmic).

Other drugs that bind albumin, particularly at site 1, are also useful in this embodiment, and can be used in drug interactions and standard pharmacy textbooks in the art (eg, “Drug Information” AHFS 1999 ( American
an Society of Health System) or "Drug
Facts and Comparisons "(published by Berney Olin and revised every four years). Assays for determining appropriate protein-biological agent combinations are disclosed therein and are used to test any combination of their ability to study using the methods of the invention. obtain.

As demonstrated below, the present invention is particularly relevant for oligonucleotide and polynucleotide therapy. The primary obstacle to gene therapy using effective antisense, anti-gene, probe diagnostics or viral or plasmid nucleotide delivery is the ability to reach the target site at a high enough concentration to achieve the therapeutic effect of the therapeutic. Because it is. The present invention is particularly useful for delivering nucleotide sequences in the form of gene therapy vectors, diagnostic nucleotide probes, or delivering nucleotide sequences in antisense or anti-genotype strategies to ultimately deliver genes in target cells. It is particularly useful for altering or detecting expression.

The oligonucleotides used in the present invention may include one or more modifications of nucleobases, sugar moieties, internucleoside phosphate linkages, or a combination of modifications at these sites. The internucleoside phosphate linkage can be, for example, a phosphorothioate, phosphoramidate, methylphosphonate, phosphorodithioate, or a combination of such similar linkages (to produce oligonucleotides modified with a mixed backbone). possible. Other oligonucleotide analogs include morpholino oligonucleotides, peptide nucleic acids, 2′-O-allyl or 2′-O-alkyl modified oligonucleotides, and N3 ′ → P5 ′ phosphoramidates. In a preferred embodiment, the oligonucleotide is a phosphorothioate oligonucleotide.

[0041] Any known method for oligonucleotide synthesis can be used to prepare the oligonucleotide. These can be any commercially available automated nucleic acid synthesizer (eg, Applied Biosystems, Inc., DNA synthesizer (M
odel 380B) and is most conveniently prepared according to the manufacturer's protocol using phosphoramidate chemistry. Phosphorothioate oligonucleotides are described in Stek and Zon (J. Chromatography,
326: 263-280) and according to Applied
Biosystems DNA Synthesizer User Bull
Letin, Models 380A-380B-381A-391-EP (1
Dec. 989) and can be purified. ODN is introduced into cells by methods known to those skilled in the art. For example, Iversen, 1991, "I
n vivo Studies with Phosphorothioate
Oligonucleotides: Pharmacokinetics P
rologue ", Anticancer Drug Des. 6: 531-8
checking ...

V. Delivery Methods In a preferred method of performing the delivery therapies of the invention, the pharmaceutical liquid agent of the invention is administered by intravenous injection, intravenous (iv infusion), transdermal. , Or intramuscularly, preferably near the target site of activity of the agent, into a mammalian host. The site of treatment may be the location of a particular tumor, the location of a particular infection, the organ that expresses a particular gene product due to differential gene activation, the site of injury or thrombus, the further progression and distribution of a therapeutic agent. Site such as a site for

A. No Ultrasound In accordance with an embodiment of the present invention, microbubbles of insoluble (eg, perfluorocarbon) gas, coated with a protein (eg, albumin), provide stable binding to oligonucleotides. It was shown to form a body (see Examples 4-6). ODN
The conjugated foam is introduced into the animal so that the protein coating
The conjugated agent can be directed to the interaction site. Eventually, the drug is released at the tissue site as foam dissipation. We have shown that the application of ultrasound is not required for targeted delivery of a biologic to the site of bioprocessing of a protein coating. This is shown in Example 9, where the ODN-conjugated microbubbles were directed to the liver and kidney and were effective in altering the metabolism of hexobarbital. This protein allows microbubbles and conjugates to pass through the processing site, and as bubbles dissipate, oligonucleotides or other biologics are released and interact at that site, and the fraction of biological agent is To achieve the same biological effect as compared to the administration of

In general, target sites are selected based on the bioprocessing of the filmogenic protein. For example, the kidney and liver take up albumin, and albumin microbubbles can be used to specifically direct administration of conjugated bioactive agents to these areas. This biodistribution is demonstrated in Example 9 as described above. Metabolism and bioprocessing of other filmogenic proteins are described in standard pharmacology textbooks (eg, “Basic and Clinical Phas”
rmology ", 7th edition, Bertram G. Katzung, Ap
pleton & Lange, 1997).

B. With Ultrasound In one embodiment of the invention, a diagnostic ultrasound field is introduced when an injected bolus reaches a target site. As demonstrated in Example 8,
Exposure of the PS-ODN-labeled perfluorocarbon-containing microbubbles to diagnostic ultrasound was performed using PS
-Does not change the strength of ODN, but releases ODN from its albumin binding.

The application of ultrasound can be achieved by various methods. For example, the ultrasound transducer can be placed directly over the target site. Conventional ultrasound devices can provide ultrasound signals from 20 kHz to several Mhz. This signal is generally applied at a level of about 3 to about 5 MHz for diagnostic ultrasound and less than 1 MHz, preferably about 20 kHz for therapeutic ultrasound. The energy provided by the ultrasound creates microbubbles that release the drug at the treatment site or into the blood pool.
The method also visualizes the bolus as it penetrates the ultrasound field.

Alternatively, an ultrasonic transducer can be placed over a site in the blood pool, allowing constant exposure of the microbubbles as they pass through the circulation. This procedure is described in Example 13
Enhances the systemic effects of certain drugs (eg, heparin), as shown in

[0048] Examples of placement sites where ultrasound augmentation can be used include kidney, ventricular (heart)
chamber, aorta or vena cava. Example 12 describes targeted sonication of the kidney in combination with delivery of labeled ODN-conjugated microbubbles. Sonication limited to one kidney resulted in a significant increase in ODN detected at this site. Further described in Section VI below is the use of targeted sonication in inhibiting arterial restenosis by administering antisense to c-myc with sonication of the affected area.

In another embodiment, the injectate can be monitored or timed until it reaches the target area. Control agents are used alone to time delivery from administration to the treatment site. After administration of the therapeutic agent, low frequency (
Ultrasound (from 20 kHz to about 2 MHz) is introduced at the target site after a suitable time interval using a suitable Doppler echo or ultrasonic echo device, so that the ultrasound field encompasses the target site, The drug is then released from the microbubbles. This point generally varies according to the organ of interest as well as the site of injection.

VI. Inhibition of Restenosis A. Background Some researchers have shown that neointimal hyperplasia occurs after vascular balloon injury as a result of smooth muscle cell migration and proliferation. (Aus
tin, G .; E. FIG. Et al., “Initial Proofation of
Smooth Muscle Cells as on Explanatio
n for Current Colony Array Sten
Osis After Percutaneous Transluminal
Coronary Angioplasty, "J Am. Coll. Car
diol. 6: 369-75, 1985; Et al., “A Casc
ade Model for Restenosis: A Special C
as of Atherosclerosis Progression ",
Circulation 86: III47-III52, 1992; Clow
es, A. W. Et al., "Regulation of Smooth Muscl
e Cell Growth in Injected Artery ", J. Am. C
ardiovasc. Pharmacol. 14: S12-S15, 1989)
. This neointima formation is associated with balloon injury and endovascular stenting.
g) plays a role in angiographic observation of restenosis after both (Mintz,
G. FIG. S. Et al., "Intravasular Ultrasound Insi
ghts into Mechanisms of Stenosis For
motion And Restenosis, "Cardiol. -Clin
. 15: 1: 17-29, 1997). Synthetic antisense oligonucleotides (ODNs), which inhibit the synthesis of the proto-oncogene responsible for vascular smooth muscle proliferation, successfully inhibited the formation of stenosis following injury to coronary arteries or carotid arteries (Shi, Y. et al.,
"Transcaterer Delivery of C-Myc Ant
isense Oligomers Reduces Neointimal
Formation in a Porcine Model of Coro
"nary Artery Balloon Injury", Circulat
ion 90: 944-51, 1994; Morishita, R .; Et al., "In
temporal Hyperplasia After Vascular Inj
urry is Inhibited by Antisense CDK2 K
inase Oligonucleotides ", J. Am. Clin. Inves
t. 93: 1458-64, 1994). Up to this point, such treatment required direct delivery intravascularly or periadientally. We have found that ODNs (specifically to c-myc and c-myb) were exposed to carbon fluoride and sonicated dextrose / albumin (PES).
DA) demonstrated binding to microbubbles (Porter, TR et al., "Int.
eraction of Diagnostic Ultrasound wi
the Synthetic Oligonucleotide-Labeled
Perfluorocarbon-Exposed Sonicated D
extrose Microbubbles, "J. Ultrasound. M
ed. 15: 577-584, 1996). Subsequent studies have shown that percutaneous low frequency ultrasound increases the deposition of ODN on blood vessels contained within fields that do not produce sound waves (Porter, TR.
Et al., "The Effect of Microbubble Gas Com.
Position and External Ultrasound Fre
quency on the Non-Invasive Enhanceme
nt of Antisense Oligonucleotide Deli
very to the Vascular Wall in Pigs ", C
irculation Suppl. 2249, 1997). The study described in Example 14 shows that c-myc bound to low frequency ultrasound and PESDA microbubbles
This enhanced vascular deposition with intravenously injected ODNs was performed to determine if it could inhibit neointima formation following balloon injury of the carotid artery.

Previous methods of delivering ODN following arterial injury in previous studies required either direct arterial delivery or peri-adeptal application (Shi et al., 1994, cited above).
Morishita, R .; Et al., 1994 (cited above); Bennett, M.
. R. Et al., "Effect of Phosphorothioated Ol"
igonucleotides on Neointimal Formati
on in the Rat Carotide Artery ", Arteri
oscler. Thromb. Vasc. Biol. 17: 2326-32, 1
997).

B. This Study Example 14 describes a pig using the transdermal ultrasound and intravenous microbubble delivery system described herein (including antisense to the c-myc proto-oncogene). Inhibition of carotid neointima formation in mice. The area across the right carotid artery did not produce sound waves before and after each injection for a total of 6 minutes, both immediately after balloon inflation and 3 days later. As demonstrated in the examples, ultrasound targeted deposition of intravenously administered ODN bound to PESDA microbubbles inhibits intimal hyperplasia, as well as significantly less intimal to lumen diameter Producing a thickness ratio inhibited stenosis formation.

In addition to being non-invasive, an ultrasound targeting approach is advantageous. Because it can be repeated at various time intervals after the injury. Peri-adventricular application of antisense to c-myc suppressed medial replication, but this suppression is no longer evidence 4 days after carotid injury in rats (Bennett MR et al.
Et al., 1997 (cited above)). In this study, as described in Example 12, a second 0.5 milligram dose of ODN conjugated to PESDA provided 3
Day after intravenous administration. Both reduced intimal hyperplasia and larger vessel lumen were observed in the ODN-PESDA group at 30 days. It is unknown whether the larger lumen size is due to vascular growth in the ODN-PESDA group or contraction in the control group. Because pre-balloon injury IVUS data was not available.

Vasodilation is an important factor in determining the degree of stenosis formation after balloon injury and has been shown to be more important than the amount of intimal hyperplasia that occurs (Ka).
kuta, T .; Et al., "Differences in Compensator"
y Vessel Enlargment, Not Initial For
, Account for Restenosis After
Angioplasty in the Hypercholesterole
mic Rabbit Model ", Circulation 89: 280.
9-15, 1994). Larger luminal area in subjects treated with ultrasound-targeted delivery indicates that a significant effect of ODN on c-myc is that inadequate compensation enlargement in response to atherosclerosis.
ent). ODN can cause lumen enlargement 1
One method is by interfering with c-myc mediated cell migration of the medium,
This is a process inhibited by the direct application of antisense to c-yc (
Bennett M. R. Et al., 1997 (cited above); Biro, S et al., "I.
nhibitory Effects of Antisense Oligo
nucleotides Targeting c-myc mRNA on
Smooth Muscle Cell Proliferation and
Migration ", Proc. Natl. Acad. Sci. USA 9
0: 654-58, 1993). Another mechanism by which ODNs can change vessel size is
By inhibiting the ability of c-myc to stimulate the production of endothelin-1 (a potent vasoconstrictor and mitogen) (Shichiri. M. et al., "Bi
phasic Regulation of the Preproendodot
helin-1 Gene by c-myc ", Endocrinology
138 (11): 4584-90, 1997).

This study confirms that in the presence of ultrasound, enhanced uptake of ODN bound to PESDA microbubbles has a significant effect on stenosis formation following carotid balloon injury and as a delivery system Demonstrate the physiological effectiveness of ultrasound and microbubbles.

Ultrasound has been shown to enhance gene expression in cultured HeLa cells when oligonucleotides are delivered to other carrier systems such as cationic liposomes (Unger, E. et al. C. et al., "Ultrasound.
Enhances Gene Expression of Liposom
al Transfection ", Invest. Radiol. 32:72
3-27, 1997). The mechanism of this enhanced cellular uptake in the presence of ultrasound has been postulated to be cavitation-induced bilayer injury of the cell membrane (Mitrago).
tri, S.M. Et al., “Transdermal Drug Delivery U
sing Low-Frequency Sonophoresis ", Pha
rmeutical Research 13: 411-20, 1996)
. Therefore, microbubbles may have inherent advantages over other carrier systems due to their ability to lower this cavitation threshold (Holland, CK et al., "
Thresholds for Transient Cavitation
Produced by Pulsed Ultrasound in a C
controlled Nuclei Environment ", J. Am. Akou
st. Soc. Am. 88: 2059-69, 1990). If cavitation is the mechanism for enhanced uptake of ODN, both the presence of microbubbles and the lower frequency ultrasound (20 kHz) used in this study may have improved uptake.

In conclusion, intravenous ODN-PESDA and percutaneous low frequency ultrasound have been shown to inhibit carotid stenosis formation in a manner similar to direct application of antisense to the vessel wall following balloon injury. I have. These data are obtained from ultrasound and O
It demonstrates that microbubble delivery systems containing DN can be a powerful non-invasive method to inhibit stenosis formation after balloon angioplasty or endovascular stenting.

[0058] (Advantages of Microbubble Preparation in VII.N ₂ depleted environment) microbubbles comprising albumin shell permit rapid diffusion of soluble gases through their membranes. Perfluorocarbon-containing microbubbles survive longer than air-containing microbubbles with the same membrane, due to the lower rate of diffusion of higher molecular weight gases and their insolubility in blood.

However, if the fluorocarbon-containing microbubbles are still prepared by sonication in air (ie, without any modification of the sonication environment), a significant amount of air gas will be generated. May be included. In this sense, "containing insoluble gas," as used herein, refers to microbubbles formed by stirring with a blood insoluble gas (eg, perfluorocarbon), but with sonication. It is also intended to include other gases present between them. Thus, microbubbles sonicated with air can be affected by concentration gradients that exist across the albumin membrane.

[0060] Mathematical models have suggested that microbubbles containing insoluble gases persist longer when tissues and blood contain nitrogen (Burkard, ME et al., "
Oxygen Transport to Tissue by Persis
tent Bubbles: Theory and Simulations "
, J. et al. Appl. Physiol. 2874-8, 1994). In the absence of blood nitrogen (ie, oxygenated blood), nitrogen from PCMB is
And reduce their size.

Thus, as shown in Example 10, PCMB exposed to 100% oxygenated arterial blood was significantly smaller in size than PCMB exposed to air blood (Table 4, Example 10). checking). This in vitro study confirms that when nitrogen diffused from the microbubbles into oxygenated blood, oxygenated blood reduced the size of perfluorocarbon-containing MBs compared to normal blood. However, oxygenated blood, as shown using albumin microbubbles containing pure air,
Perfluorocarbon-containing microbubbles were not completely destroyed due to the rapid diffusion of soluble gas from the microbubbles (Wible J. Jr. et al., "Effects").
of Inspired Gas on the Efficiency of A
Ibunex® in Dogs ”, Circulation 88.
(Supplement): 1-401 (1993)).

Since the surface tension and the absorption pressure increase as the diameter of the microbubbles decreases,
It was hypothesized that the image intensity produced by intravenous PCMB was also affected by changes in nitrogen and oxygen concentrations inside and outside the microbubbles. By increasing the microbubble oxygen content (thus, by lowering the partial pressure of N ₂ in the micro in bubbles), it was hypothesized that residual microbubbles in the blood may be extended. This is accomplished by forming the microbubbles in an environment free of N ₂ depleted environment or N _2.

[0063] As used herein, the term "N ₂ depletion" or "nitrogen depletion" refers to less N ₂ content than the N ₂ content of the air. As a result, the partial pressure of N ₂ in the micro in bubbles filled with gas formed by sonication in the presence of such an environment, min N ₂ achieved by sonication in the presence of air Lower than pressure. (Air typically contains about 75.5% by weight and about 78% by volume nitrogen). Typically, N ₂ depletion environment has a higher oxygen content than the oxygen content of the air. N ₂ depletion environment, if it is substantially free of N ₂ (e.g., pure oxygen supplied commercially), "free of N _2."

[0064] Therefore, the microbubbles, typically gas containing no N ₂ depleted gas or N ₂ (e.g., oxygen) to, by blowing the interface between the sonicating horn and the solution, N ₂ in an environment containing no depletion environment or N ₂ is sonicated. In this case, "insoluble gas-containing" microbubbles, insoluble gas (e.g., perfluorocarbon) contains, and it is expected to contain some quantity of gas containing no N ₂ depleted gas or N _2. (Some small quantity of air, but may enter the microbubbles even under these conditions, the gas contained in the microbubbles. Regarded as substantially not including N ₂₎ This modification is substantially smaller It has been found to create microbubbles. The microbubbles were more stable in the bloodstream, resulting in improved contrast and drug delivery. Such effects were consistently observed in the non-thoracotomy study described in Example 11 and resulted in greater myocardial contrast than PCMB sonicated in the presence of air. Even with intermittent imaging using short pulse interval of up to 100 milliseconds (10 Hertz imaging), the contrast of the visually apparent myocardium was sonicated in an environment free of N ₂ It was still achieved with microbubbles.

The present invention includes the use of such contrast agents in pharmaceutical compositions and the application of ultrasound as a targeted delivery system. Environment free of N ₂ depleted environment or N ₂ in the preparation of contrast agents is also an improvement of the effect of contrast agent in imaging of the myocardium.

EXAMPLES The following examples are for illustrative purposes only and are not intended to limit the invention in any way. It will be appreciated by those skilled in the art that numerous other combinations of protein bioactive agents can be used in the present invention and are contemplated herein. For example, filmogen protein (filmogenic p)
When the protein is an α1 acid glycoprotein, the bioactive agent can be lidocaine, indelal, or heparin.

In all the examples below, all parts and percentages are by weight unless otherwise specified, and all dilutions are by volume.

Example 1 Phosphorothioate Oligonucleotide Synthesis Chain elongation synthesis was performed using an ABI Model 391 DNA synthesizer (Perk).
in Elmer, Foster City, Calif.) or on Lynx Therapeutics, Ind.
c. (Hayward CA). 1 μmol synthesis is ABI
With user bulletin 58, a sulfur treatment using cyanoethyl phosphoramidite and tetraethylthiuram disulfide was used. The radiolabeled oligonucleotide was synthesized as a source of hydrogen phosphite by Glen Research (Bethesda, MD).

Example 2. Preparation of a microbubble suspension 5% human serum albumin and 5% dextrose were obtained from commercial sources. 3 parts of 5% dextrose solution and 1 part of 5% human serum albumin solution
Aliquots (16 ml total) were drawn up into a 35 ml Monojet® syringe. Each dextrose / albumin sample was manually agitated with 8 ± 2 ml of either decafluorobutane or air, and the sample was
Exposure to 80 ± 5 seconds of electromechanical sonication at kilohertz. The average size of a sample of four consecutive, perfluorocarbon exposed and sonicated dextrose / albumin (PESDA) microbubbles made in this manner was 4.6 ± 0. 4 microns. The average concentration is then 1.4 × 10 ⁹ bubbles / as measured by a Coulter counter.
mL.

Example 3 Preparation of Microbubble / ODN Conjugate Sequence 5′-TAT GCT GTG CCG GGG TCT TCG GG
C3 '(24mer complementary to c-myb) (SEQ ID NO: 2) and 5'TTAG
A homogenous ³⁵ S-labeled PS-ODN (phosphorothioate oligonucleotide) having GG (SEQ ID NO: 3) was treated with a sonicated dextrose / albumin (PESDA) microbubble sample solution exposed to perfluorocarbon. .5
Incubated for 30 minutes at 37 ° C. in a final volume of ml. The solution was set up so that microbubbles could form at the top; samples could then be removed from either the bottom clear solution or the top layer containing microbubbles.

Example 4. Phosphorothioate ODN binding to washed or unwashed PESDA microbubbles To make washed microbubbles, a solution of the microbubbles as prepared in Example 1 Washing with a 1000-fold excess of% dextrose removed albumin not bound to microbubbles. Microbubbles were generated for 4 hours. The lower solution was then removed, leaving the washed foam, and then mixed with 0.9% sodium chloride. The albumin protein concentration in the washed microbubbles can be determined by the Bradford assay (Bradford, M. et al., "A Rapid and Sensitive".
Method for the Quantification of Mi
program Quantities of Protein Utility
ing the Principle of Protein-Dye Bin
ding ", Anal. Biochem. 72: 248, 1976) was 0.28 ± 0.04 mg / ml.

The radiolabeled 24-mer PS-ODN was added to a suspension of washed or unwashed PESDA microbubbles at a concentration of 5 nM. The radioactivity in the solution was measured using a liquid scintillation counter (model LSC7500; Beckman).
(Instruments GmbH, Munich, Germany). Hydrocount Biodegradable Scintillation Cocktail (5ml)
Was added to the 100 μl sample. Samples were counted immediately after each experiment and then again after 24 hours to reduce the effects of chemiluminescence and quenching.

PESDA of PS-ODN, counted by liquid scintillation counting
The distribution to microbubbles (top layer), washed lower layer (without albumin) and unwashed lower layer (with albumin) is shown in Table 1. The recovery of total radioactivity in the experiments reported in Table 1 was about 96%.

[0074]

[Table 1] This data shows that albumin in unwashed solution without microbubbles
It shows that it binds to PS-ODN. Removal of albumin without microbubbles (washed foam) resulted in an increase in the distribution of PS-ODN with PESDA microbubbles (ratio:
1.67 for TTAGGG PS-ODN, and c-myb PS-OD
N is 2.16).

Example 5 Binding Affinity of ODN to Washed Albumin-Coated Microbubbles To assess the binding affinity of PS-ODN to the washed microbubbles of Example 4, excess as a competitive ligand for the binding site A mixture containing an increased amount of non-radioactive PS-ODN was prepared. Sequence 5′-d (CCC TGC TCC CCC
Non-radioactive PS-OD with (CTG GCT CC) -3 '(SEQ ID NO: 4)
N 20mer was administered in a series of increasing concentrations (0, 3.3, 10, 32.7, 94.
5, 167, and 626 μM) were added to tubes containing radioactive 24mer. Each foam suspension was mixed by inversion and incubated at 37 ° C. for 60 minutes.

FIG. 1 shows the observed binding data as a Lineweaver-Burke plot.
Shown in The equilibrium dissociation constant for binding to microbubbles, K _m (calculated for seven concentrations performed in duplicate) was 1.76 × 10 ⁻⁵ M.

Example 6 ODN-Microbubble Competitive Binding Study Three groups of samples were prepared in triplicate as follows, and each sample was incubated for 20 minutes at room temperature.

Group A (control): 100 μl microbubbles / 900 μl saline + 2 μl unlabeled 20mer Group B: 100 μl microbubbles / 900 μl saline + 2 μl FIT
C-labeled 20mer (final 20mer concentration = 151 nM) Group C: 100 μl microbubbles / 800 μL saline + 2 μL FIT
C-labeled 20mer + 100 μL unlabeled 20mer.

The flow cytometry analysis was performed using a 100 mW air-cooled argon laser and Lys
FACStar Plus with isII collection and analysis software (Be
cton Dickinson). Using the list mode data for collected microbubbles minimum 10 ^4, and analyzed independent for each sample. The distribution of FITC-labeled microbubbles is provided in Table 2.

[0080]

[Table 2] PE = percent event; MI = mean intensity; SE = standard error ¹ mean is significantly different from control; P <0.0012 ² mean is significantly different from 151 nM sample; P <0.001.

The significant decrease in mean fluorescence intensity in samples containing excess unlabeled PS-ODN
Shows that binding to microbubbles can be saturated. As a result, PS-O with the microbubble surface
Nonspecific interactions of DN are limited.

Example 7. Binding of ODN to microbubbles containing air vs. binding of ODN to microbubbles containing perfluorocarbons PS-O to sonicated dextrose / albumin microbubbles containing air
The uniformity of DN binding versus the uniformity of PS-ODN binding to sonicated dextrose / albumin microbubbles containing perfluorocarbon was measured by flow cytometry. Gaussian distribution of PS-ODN on washed PESDA microbubbles (data not shown) indicated that the albumin of these microbubbles retained their binding site for the oligonucleotide. The absence of a Gaussian distribution for the washed RA-SDA microbubbles indicated that loss of albumin binding site 1 for this oligonucleotide occurred during sonication of these microbubbles. (For albumin binding properties, especially when they relate to oligonucleotides, see Kumar et al., "Characterization of Bind."
ing Sites, Extent of Binding, and Drug
Interactions of Oligonucleotides wi
th Album "Antisense Res. Dev. 5: 131-1
39 (1995)).

From the above, it can be observed that PS-ODN binds to albumin in PESDA microbubbles, indicating that binding site 1 on albumin is responsible for the production of these bubbles by electromechanical sonication. It is biologically active after birth. However, this binding site affinity is lost if electromechanical sonication is performed using only air. Furthermore, removal of albumin without PESDA microbubbles by washing results in significant distribution of PS-ODN with microbubbles (Table 1). These observations demonstrate that albumin denaturation does not occur with perfluorocarbon-containing dextrose / albumin solution during sonication, as suggested by sonication in the presence of air.

The retained biological activity of albumin (especially at site 1) in the PESDA microbubbles was washed in the presence of an increased amount of excess non-radioactive PS-ODN to compete with the ligand for the binding site. Was confirmed by the affinity of PS-ODN binding to the resulting PESDA microbubbles (Table 2). A significant decrease in average fluorescence intensity in samples containing excess unlabeled PS-ODN indicates that binding to microbubbles can be saturated.

Example 8 Ultrasonic Exposure of Microbubbles with Bound Oligonucleotides As described previously (Mor-Avi, V. et al., “Stability o.
f Albunex® Microspheres under U
itrasonic Irradiation; an in vitro St
udy "J. Am. Soc. Echocardiology 7: S29, 19
A variable flow microsphere scanning chamber similar to 94) was used. This system is based on the Masterflex® flow system (Micro
gon, Inc. , Laguna Hills, CA). The scanning chamber is surrounded on each side by a chamber filled with water and bound on each side by an acoustically transparent material.
PS-ODN labeled PESDA microbubbles (0.1 ml) were injected as a bolus proximal to the scanning chamber over 1 second and then controlled at 100 ml / min into a tap water-filled scanning chamber. Flowed through the plastic tubing at a flow rate. As the foam passed through the scanning chamber, the scanner (2.0 MHz frequency, 1.2 MPa peak negative pressure) was set to deliver ultrasound at a conventional 30 Hz frame rate. A control run where no ultrasound was delivered was also performed.

After passing through the scanning chamber, the solution was passed through a cylinder with memory via plastic tubing of the same size. The first 10 mL was discarded. The next 10 mL was collected and left to separate the top microbubbles from any free oligonucleotide present in the lower part of the sample.

Drops from both the upper and lower runs of the eluate were placed on hemacytometer slides and analyzed using a 10 × magnification. Photos of these slides were made and the number of microbubbles was manually counted over a 36 square centimeter field of view. The higher and lower portions of the eluate were also mixed 15 times (v / v) with a solution of formamide and EDTA and heated at 95 ° C. for 5 minutes. These samples were then run on an Applied Biosystems M
odel 373A DNA sequencer and tested on a 20% polyacrylamide gel. Data was obtained using GeneScanner® software and the area of fluorescence intensity under the curve could be measured.

[0088]

[Table 3] ^a Top = top layer of eluate after sonication; bottom = bottom layer after sonication There is significant loss of microbubbles in the top layer after exposure to ultrasound (219 without ultrasound). For ± 54 microbubbles, 53 ± 21 microbubbles in the upper layer of the eluate when exposed to diagnostic ultrasound). This loss of microbubble count was evident regardless of the initial PS-ODN concentration in the upper foam-containing layer. The PS-ODN concentration in the lower layer without bubbles was significantly increased as measured by gel electrophoresis. This data shows that exposure of PS-ODN-labeled PESDA microbubbles to diagnostic ultrasound does not change the integrity of PS-ODN but releases it from its albumin binding.

Example 9 Altered Biodistribution via Microbubble Delivery of Antisense Oligo Antisense phosphorothioate oligonucleotides to the cytochrome P450 IIB1 gene sequence were designed to alter hexobarbital metabolism (PB is CYP Stimulates IIB1 mRNA and consequently hexobarbital, which is hydroxylated by CYP IIB1, is metabolized more rapidly and its calming effect is reduced. Is expected to react).

An oligonucleotide was synthesized according to the known sequence of rat cytochrome P450 IIB1, and the oligonucleotide had the following sequence: GGA
GCA AGA TAC TGG GCT CCAT (SEQ ID NO: 5) and AAA GAA GAG AGA GAG CAG GGA G (SEQ ID NO: 6)
. These oligos were conjugated to dextrose / albumin microbubbles exposed to perfluorocarbon and sonicated (PESDA) as described earlier and delivered intravenously to rats. Male Sp between 210-290 grams
Rague-Dawley rats (Sasco, Omaha) were used for all studies. These were raised in the animal area of the University of Nebraska Medical Center, an AAALAC certified animal resource facility. The animals were exposed to a 12 hour light / dark cycle and Purina rat chow and tap water were available ad libitum.

[0091] Rats in designated groups were injected intraperitoneally with phenobarbital (PB) (Mallinckrodt, St. Louis) at 80 mg / kg / day for 2 days. PB injection was given at the same time as ODN microbubble injection. Phosphorothioate OD
N infusion was at 1 mg / kg / day for 2 days. 48 hours after the first infusion, sleep time was measured.

Each rat received 100 mg / kg of hexobarbital (Sigma, St.
. Louis) was injected intraperitoneally at the end of the second day of treatment with PB and / or ODN. The animals are placed on their backs, ensuring that they are still under sedation from hexobarbital; and sleep time is reduced so that they are placed on their backs until they wake up It is defined as the time to be.

The results show that delivery of the oligonucleotide-conjugated microbubbles greatly improved the efficacy of the drug. Control rats had a sleep time of about 23 minutes. P
Administration of B reduced sleep time to 11.4 ± 4.5 minutes. However, 1/2
Rats that received zero dose of microbubble conjugated oligos experienced more than 50 minutes of sleep. By comparison, non-microbubble conjugated oligos did not significantly alter sleep time (about 13 minutes).

To determine the biodistribution of oligos, animals were finally sacrificed with ethyl ether and microsomes were removed from Franklin and Estabbrook (1
971). Livers were perfused through the portal vein with 12 ml of 4% saline and then removed from the animals. Chop the liver,
Homogenize in 0.25M sucrose (Sigma) and Sorvall
8000 in RC2-B centrifuge tubes (Dupont, Wilmington, DE)
× g, and centrifuged at 4 ° C. for 20 minutes. The supernatant is collected, resuspended in 0.25 M sucrose, and placed in a Sorvall OTD55B ultracentrifuge tube (Dupont) for 1 hour.
Centrifugation was performed at 00,000 × g and 4 ° C. for 45 minutes. 1.15% K pellet
Resuspended in Cl (Sigma) and centrifuged at 100,000 xg for 1 hour at 4 ° C. The final pellet was resuspended in an equal volume of buffer (10 mM Tris-acetic acid, 1 mM EDTA, 20% glycerol; Sigma) and frozen at -80 ° C.

The protein concentration was determined by the Bradford assay (Bradford, 1976).
). Aliquots (80 μl) of the homogenate were transferred to 96-well plates (Becton, Dickinson Labware, Lincoln).
Park, NJ). Then, Bradford reagent (20 μl; B
io-Rad, Richmond, CA) and add the plate to a microplate reader (Molecular Devices, Newport M).
N) at 595 nm. The data was compared to a standard curve generated using known bovine serum albumin (Sigma).

[0096] The CYP IIB1 content is determined by pentoxyresorufin.
rufin) O-dealkylation (PROD) activity (eg,
Lubet, R .; A. Arch. Biochem. Biophys. 238
(1): 43-8, 1985). For microsomal samples, 1 ml of 0.
1 mg protein in 1 M potassium phosphate buffer, 1 ml 2 μM 5-pentoxyresorufin (Pierce, Rockford, IL), and 17 μl
Of 60 mM NADPH was mixed and incubated at 37 ° C. for 10 minutes. The mixture was then added to a 2 ml cuvette and read on an RF5000U spectrofluorimeter (Shimadzu, Columbia, MD) using an excitation wavelength of 530 nm and an emission wavelength of 585 nm. Determine the concentration of the unknown sample
Calculated from a standard curve of resorufin (Pierce, Rockford, IL). The results were recorded in nmol resorufin / mg protein / min.

Direct measurement of the CYP IIB1 protein was measured by an ELISA assay using antibodies directed against the CYP IB1 protein (Schuurs and Van Weeman, Clin. Chim. Acta 81 (1): 1-
40, 1977). Liver samples (50 μg per well) were transferred to 96-well nu
1 on nc-immuno plate (InterMed, Skokie, IL)
Plated overnight in 00 μl of 0.35% sodium bicarbonate buffer.
Microsomes were washed three times with 1% bovine serum albumin in PBS (PBS / BSA) and incubated for 1 hour at 37 ° C. with 200 μl of PBS / BSA. The PBS / BSA was removed and 50 μl of CYP IIB1 antibody (O
xygene, Dallas, Tex.) and incubated at 37 ° C. for 1 hour. The microsomes were transferred to saline / Tween® 20 (S
igma) and washed with 50 μl of horseradish peroxidase antibody (
Bio-Rad) was added. The microsomes were incubated at 37 ° C. for 1 hour, washed 5 times with saline / Tween®, and twice with 85% saline. Horseradish peroxidase substrate (100 μl; Kir
kegaard & Perry Labs, Gaithersburg, Md.) and add the plate to a microplate reader (Molecular).
Devices) at 405 nm for one hour. The result is
D / min was recorded as horseradish peroxidase activity.

The results demonstrated that the oligoconjugated microbubbles directed the oligo to the liver and kidney, sites of phenobarbital metabolism.

Example 10. Size and concentration of PESDA microbubbles after sonication in normal and oxygen-enriched arterial blood. Arterial blood was obtained from 4 dogs and 3 healthy pigs immediately before sacrifice. Collected under air inhalation conditions. In four of the animals, additional arterial blood was
Obtained after inhalation of 0% oxygen for a minimum of 10 minutes. The blood was collected in a 60 ml heparinized syringe and kept at 37 ° C. in a warm water bath until injected into the scanning chamber. Immediately before injection of blood into the scanning chamber,
0.2 ml of perfluorocarbon-containing microbubbles (PCMB) was injected into the blood in a 60 ml syringe through a stopper and mixed by gently inverting and rotating the syringe by hand.

The sample was then injected into the scanning chamber at a flow rate of 50 ml / min. Once the chamber is filled, open the closed stopcock connecting the scanning chamber to the plastic tubing used for injection, and expose with ultrasound (1 Hz or conventional 30-45 Hz frame rate). Started intermittently). After ultrasonic exposure, the shed blood flowed out of the scanning chamber to tubing connected to a graduated cylinder. First 10
The ml of blood was discarded and the next 15 ml of blood was collected as three 5 ml aliquots into an inverted and capped syringe. Three minutes after collection of the last 5 ml sample, the tuberculin syringe was immersed in the highest level of eluted blood and one drop was placed on a hemocytometer slide; select this length of time to elute Microbubbles in the blood rose to the top and allowed to be collected. Then, the hemocytometer slide was subjected to an optical microscope (Olympus BH-2, Olly) at a magnification of 40 times.
mpus America Inc. , Woodbury, NY), and the field containing the highest concentration of microbubbles was imaged with a hemocytometer field.

The photograph was later magnified and a 25 cm ² field was selected to analyze the microbubble concentration and mean diameter in this field. The concentration was determined by counting the total number of vesicles across the slide. The microbubble concentration measured using this technique was Cou
It correlates very closely with the measurements in the liter counter and the size measurements in this technique are based on the known 5 micron spheres (Coulter Size Sta).
ndards Nominal 5 μm Microspheres, Miami
i, FL).

(In Vitro Scanning Chamber :) The scanning chamber system is a peristaltic Masterflex flow system (Microgon, Inc.,
Laguna Hills, Calif.). Sealed on both sides of the scanning chamber is a cylindrical saline-filled chamber bound by a 6.6 micron thick acoustically transparent latex material (Safeskin, Inc .; Boca Rato).
n, FL). The pressure in the scanning chamber during the ultrasonic exposure is measured by a pressure transducer (model 78304A; Hewlett) located proximal to the scanning chamber.
Packard Co. , Andover, Mass.) And averaged 7 ± 3 mmHg throughout all trials.

Two different 2.0 MHz ultrasound transducers were used for in vitro studies (Hewlett Packard 1500; Andover, Mass.)
sachusetts; and HDI 3000, Advanced Tech.
nology Laboratories, Bothell, Washington
on). The peak negative pressure generated is 1.1 MPa for the Hewlett Packard transducer and 0 for the HDI 3000 scanner.
. It was 9 MPa. The imaging depth for all studies was 8.2 centimeters, and the focus for both transducers was 8 centimeters. The frame rate for each converter can be conventional (30-42 Hz) or intermittent (1
Hz). All images from the scanning chamber were recorded on high fidelity videotape.

Results: Table 3 demonstrates the difference in average microbubble size for PCMB after exposure to ultrasound in arterial blood (air and 100% oxygen). When PCMB is exposed to 100% oxygenated arterial blood, insonation
There was a significant decrease in the average microbubble size after (t =) (p = 0.01). Smaller microbubble sizes were observed after intermittent imaging (7.3 ± 3.7 μm air vs. 6.
4 ± 3.2 μm 100% oxygen) and after conventional imaging (7.5 ± 3.5 μm air versus 6.3 ± 3.0 μm 100% oxygen).

Microbubble concentrations were significantly reduced after exposure to conventional frame rates when compared to intermittent imaging in pulmonary arterial blood (Table 3). However, conventional frame rates did not destroy as many PCMBs as did in oxygenated arterial blood at the same transducer output.

[0106]

[Table 4] Microbubble concentration decreased after exposure to conventional frame rates when compared to intermittent imaging in air arterial blood (Table 4). However, the conventional frame rate is
At the same transducer output, more PCMB than destroyed in oxygenated arterial blood
Did not destroy.

Example 11 N_TwoA microbubble formed in an environment that does not contain air formed in an air environment
Vivo microbubble imaging study) Perfluorocarbon-containing microbubbles (PCMB) were prepared as described in Example 1.
Made. 1 part of 5% human serum albumin and 3 parts of 5% dextrose (1 in total)
6 ml) was manually stirred with 8 ml of decafluorobutane. After stirring,
The samples were subjected to electromechanical sonication for 80 ± 2 seconds. Ultrasonic treatment for 80 seconds
The process was performed in two different environments: air (RA) or 100% oxygen (N _Two Sonication horn and perfluorocarbon
The interface between the dextrose / albumin solution was blown during sonication.

(In Vivo Imaging :) Microbubbles formed by sonication of an aqueous protein solution are injected into a mammal to alter the acoustic properties of a given area, which is then sonicated. Diagnostic ultrasound imaging methods are known which are scanned by a medical device to obtain images for use in medical procedures. For example, U.S. Pat.
See 572,203, 4,718,433 and 4,774,958.

During sonication, 100% oxygen (O ₂ PCMB) or air (RA PCM)
The microbubble samples exposed to either of B) were injected intravenously into dogs. Imaging was performed using a 1.7 MHz harmonic converter (HDI 3000; Advanced Tec).
hnology Laboratories; Bothell, Washing
ton). The transducer output was set to 0.3-0.8 MPa and the video intensity from two different microbubble samples
y) kept constant for all comparisons. The frame rate for comparison of myocardial video intensity minus background is 43 Hz (conventional) or 10 Hz (
Intermittent). All procedures are institutional A
approved by the National Care and Use Committee, and the Position of the American Heart A
According to Sociation on Research Animal Use.

A bolus injection of RA (air) PCMB and O ₂ PCMB was 0.0025
ml / kg or 0.005 ml / kg. The concentration of microbubbles in the sample was determined to be the same. Peak front and back wall video intensities were determined using a Tom-Tech review station (Louisville, C.).
digitalized Super VH obtained offline using the
Measured from S-videotape images (Maxell, Japan). Tom-Te
The ch review station quantifies the video intensity over a gray scale range of 1-255. The area of interest was located at the center of the myocardium in each segment.

In addition to this quantitative analysis, a visual assessment of local myocardial contrast enhancement in the anterior, septum, sidewall, and posterior regions from the short axis visual field was performed by two independent investigators. Was. Each region was assigned a 0 (no myocardial contrast), 1+ (medium myocardial contrast enhancement) or 2+ (bright myocardial contrast enhancement approaching cavity intensity).

A total of 6 comparisons of peak myocardial video intensity between O ₂ PAMB and RA PCMB were performed in three dogs. In Table 5, it can be seen that PCMB sonicated in the presence of 100% oxygen prior to injection was similar in size and concentration to PCMB sonicated in the presence of air. However, in all three dogs,
Peak myocardial video intensity using a 10 Hertz frame rate (intermittent imaging) was significantly higher for PCMB sonicated in the presence of 100% oxygen.

Only oxygenated PCMB is consistent with the dose used for transthoracic imaging.
Resulted in a homogeneous myocardial contrast. Visual myocardial contrast is RA PCM
Intravenous O as compared to 2+ in 9 out of 24 areas after the same dose of B _Two 2+ in 20 out of 24 regions after PCMB injection (p = 0.001)
.

[0114]

[Table 5] Only oxygenated PCMB is consistent with the dose used for transthoracic imaging.
Resulted in a homogeneous myocardial contrast. Visual myocardial contrast is RA PCM
Intravenous O as compared to 2+ in 9 out of 24 areas after the same dose of B _Two 2+ in 20 out of 24 regions after PCMB injection (p = 0.001)
.

As expected, microbubble concentrations in air blood were significantly reduced when exposed to higher frame rates. However, this destruction with a faster frame rate was somewhat attenuated when PCMB was in oxygenated blood. The reason for this difference is not clear. One possibility is that faster diffusion of nitrogen from microbubbles in oxygenated blood results in higher internal concentrations of perfluorocarbons,
Thus, the diffusion gradient for the insoluble perfluorocarbon was increased. Due to its low solubility, its enhanced diffusion from microbubbles leads to the formation of smaller, non-encapsulated perfluorocarbon microbubbles. The resolution of the hemocytometer cannot distinguish between unencapsulated microbubbles and encapsulated microbubbles, and therefore calculates them both. This explanation may also explain the smaller average microbubble size observed for PCMB exposed to 100% oxygenated arterial blood.

Statistical analysis: Independent t-test was used to compare the size and concentration of microbubbles in PCMB samples exposed to different gases during sonication. Using this again,
Differences in peak myocardial video intensity in in vivo studies were compared. If the data was not normally distributed, a non-parametric test was performed. Intravenous O ₂ PCMB
Comparison of visual myocardial contrast enhancement after and RA PCMB is shown in the contingency table (co
This was performed using a ninency table (Fisher's exact test).
A p-value lower than 0.05 was considered significant.

The coefficient of variation was used to measure interobserver variability in measuring microbubble size and concentration in in vitro studies. Independent inter-observer differences were used to compare interobserver variability in peak myocardial video intensity.

[0118] Two independent observers are operated either intermittently or conventionally with an ultrasound framer.
The size and concentration of microbubbles on six different slides exposed to the slides were measured.
Size of microbubble size by 2 s independent observers in 6 different samples
The coefficient of variation for the measured value was 8% (r = 0.95; p = 0.004).
On the other hand, the coefficient of variation for the independent measurement of microbubble concentration is 9% (r = 0.99; p
<0.001). Two independent investigators on transthoracic imaging
The average difference reported in the peak myocardial video intensity measurements was 4 ± 4 units (r = 0
. 94, SEE = 5 units; p <0.001; n = 24 comparisons) _Two 16 in the anterior wall peak myocardial video intensity between PCMB and RA PCMB
Than the mean difference of units and the mean difference of 13 units in posterior wall peak myocardial video intensity
Low enough. Two investigators found that in 37 of the 44 areas (84%)
Consistent in visual degree of trust enhancement. The five discrepancies are RA PCMB
Was in the visual classification of myocardial contrast enhancement (in two areas
0 vs. 1+ vs. 3+ in 3 regions. Whether 1+ or 2+ exists
The three areas where there was a mismatch for were assigned 2+ in the statistical analysis
.

Example 12. Effect of targeted sonication on uptake of ODN from microbubbles. In vivo study of the effect of sonication on renal uptake of PS-ODN conjugated microbubbles. Performed in three dogs. Fluorescently labeled PS-ODN PE
An intravenous injection of SDA microbubbles (0.2 ml) was given in the femoral vein. At the same time, the left kidney was externally placed with a diagnostic ultrasonic transducer of 2.0 MHz to 2.5 MHz (
The peak negative pressure is 1.1 MPa), and the insonify
)did. The kidney was insonified with a 30 Hz frame rate for a minimum of 2 minutes after injection and during the appearance of visually apparent contrast in the kidney. In each dog, left ventricular and pulmonary artery pressures were monitored before and after renal injection using saline-filled catheters placed in the left ventricle and embryonic artery, respectively. Approximately 4 hours after injection, the dog was sacrificed and both kidneys were removed. Sections were obtained from the renal cortex and sampled for PS-ODN and counted using the gene scanning method described above (Example 8). Histological sections were also obtained for glomerular and nephron analysis for fluorescence.

In one dog, up to 10-fold greater uptake of PS-ODN was present in the insonified kidney compared to the non-insonified kidney after intravenous PS-ODN in labeled PESDA. In two of the three dogs, PS-
Partitioning of ODN uptake into the insonified kidney was evident. In the first dog, P in the insonified kidney was compared to non-insonified kidney.
There was a 3-fold higher uptake of S-ODN. In the second dog, there was over 9-fold higher uptake of PS-ODN in the insonified kidney. But,
In the third dog, there was no difference in PS-ODN uptake between the two kidneys. After intravenous injection of PESDA microbubbles labeled with PS-ODN, there were no hemodynamic changes. Histological examination of the postmortem kidney is also possible by diagnostic ultrasound.
There was no destruction of any glomeruli or tubular cells.

Example 13. Effect of sonication on heparin uptake from microbubbles Intravenous heparin was administered to dog subjects in a bolus dose of 1500 units in three different settings. In setting 1, heparin was given as free drug. Setting 2
Provided 1500 units of heparin conjugated to orosomucoid PESDA, but no ultrasound was applied to the blood pool. In setting 3, Orosomucoid PE
The same dose of heparin conjugated to SDA (1500 units) was given and ultrasound was applied to the blood pool. A baseline measurement of activated partial thromboplastin time (PTT) was measured and then repeated at 5 minute intervals after each injection for a minimum of 15 minutes.

In setting 1, the PTT increased by more than 106 seconds at 5 minutes, but increased by 30 minutes at 15 minutes.
It returned to ~ 60 seconds. At setting 2, the PTT reached over 106 seconds after 10 minutes and returned to 60-80 seconds at 15 minutes. At setting 3, the PTT is 1 after 15 minutes.
Remained for more than 06 seconds; no further measurements were taken.

Example 14. Inhibition of Carotid Neointima Formation Using an Intravenous Microbubble Delivery System Including Intravenous Ultrasound and Antisense to the c-myc Proto-Oncogene All procedures were performed using the Institutional Animal Care an
d. Approved by the Use Committee and Position
of the American Heart Association on
According to Research Animal Use. Twenty-eight domestic pigs were premedicated with aspirin (325 mg PO). Then, ketamine (20 mg / k
g), general anesthesia was administered using xylazine (4 mg / kg) and supplemental pentobarbital. The pig was intubated and placed in the respiratory air. S
A wan Ganz catheter was advanced into the pulmonary artery to monitor pulmonary pressure. Intravenous heparin (4,000-5,000 units), atropine (0.5-
1.0 mg), and sublingual nifedipine (10-30 mg). An 8F guide catheter was placed in the proximal portion of the right carotid artery. Too large balloon (6.0
The artery was injured by dilating the blood vessels at an average of 107 ± 34 seconds (range 90-240 seconds) at mm-10.5 mm). The intervention was performed by an experimental interventional cardiologist (UD) who did not know which treatment the pig would subsequently receive.
Vessel patency after injury was determined by Hexabrix or Renographin-76.
Was confirmed by angiography using intra-carotid injection.

The first 20 animals were randomized and received one of three treatments following balloon injury: (a) Intravenous phosphorothioate O for c-myc bound to PESDA
DN (0.5 mg; Lynx Therapeutics; Haywar
d, California) (ODN-PESDA); (b) Same dose of intravenous antisense to c-myc alone (ODN alone)
Or (c) no injection (control).

Injections were repeated 3 days after balloon injury in pigs with ODN-PESDA and ODN alone. All animals received ketorolac (60 mg) and Solumedrol (40 mg) intravenously to prevent a pulmonary hypertension response in pigs randomized to receive microbubbles.

The last eight pigs received different ODN for c-myc (Morpholino; AVI Biopharma, Inc .; Corv
allis, Oregon). In these pigs, after balloon injury, the first four received ODN conjugated to PESDA, and the last four received ODN alone (n
= 2) or no treatment (n = 2).

In animals randomized to ODN-PESDA, 50 watts / cm ²
Percutaneous 20 KHz ultrasonic probe with the output of (Model XL2020; He
at Systems; Farmingdale, New York) before and after each injection, both immediately after balloon inflation and on day 3.
The area above the right carotid artery was insonified for a minute. To avoid skin irritation due to heating of the probe, a 1.5-2.0 cm coupling gel was placed between the skin surface and the probe tip using an inverted 12 ml syringe that was inverted. . The position of the right carotid artery is determined using a diagnostic transducer (HDI3000; Advan).
ced Technology Laboratory; Bothell, Wa
(Shington).

(Measurement 30 days after balloon injury) 30 days after balloon injury, pigs were sacrificed after performing intravascular ultrasound (IVUS) measurements. 2.9 or 3.1 F 3
0 Megahertz IVUS catheter (CVIS; Sunnyvale
, CA) was advanced into the distal carotid artery under a fluorescence microscope and a motorized retraction of the catheter was performed. Lumen area, total blood vessel area (lumen + any angiogenic plaque),
Off-line area measurements of vessel diameters were made at sites of largest plaque and smallest lumen in previously injured areas. The pig was then sacrificed and serial sections of the carotid artery were made after fixation. Digital area measurement (NI
H Image 1.61; Bethesda, Maryland). Since this vessel did not undergo perfusion fixation, the value of maximum endothelial thickness was IV
The vessel diameter at the site of balloon injury measured by the US was indexed. IVU
Both S and histological measurements were made by investigators (WH and SR, respectively) who did not know which treatment regimen this pig received.

Statistical analysis: Differences in IVUS measurements of total vascular area, lumen area, and lumen diameter at the site of balloon injury, and histological measurements of maximum endothelial thickness in the three groups were analyzed by analysis of variance (Student-Newmann-Keuls multiple comparison procedure). Since IVUS could not be performed in three pigs that received ODN alone, data from ODN alone and controls were also combined and Student t-test or Mann-Whitney Rank
Compared to ODN-PESDA by Sum test. Interobserver variability in IVUS and histological measurements was calculated by having the same investigator repeat the measurements at different time points and calculate the percent difference between the measurements.

(Results) Two pigs died during the balloon injury protocol. Five pigs (three ODN-PESDA, one IV ODN alone, and one control) had no histological evidence of injury or endothelial hyperplasia 30 days after injury. Of the remaining 21 pigs, 8 received ODN-PESDA, 7 received intravenous ODN alone, and 6 received none. Pigs treated with 20 KHz ultrasound were superficial at the site of the applied ultrasound.
This surface delamination was no longer evident in the 30-day trace.

Table 1 contains intravascular ultrasound data and histological data in the three groups.
Intravascular ultrasound was possible in 16 pigs. In three of the pigs that received ODN alone, this could not be done because of thrombotic occlusion 30 days after injury, and in one pig each receiving ODN-PESDA and ODN alone, For technical reasons this could not be done.

The total vascular area and luminal area at the site of injury, as measured by IVUS, was
It was significantly greater in the SDA group. This larger vessel size was seen despite a significantly smaller maximum endothelial thickness in the same group in histology. When histological measurements of maximum endothelial thickness indexed IVUS vessel diameter, there was a clear distinction between the three groups (Table 1).

The control pigs had smaller blood vessels at 30 days, but greater inner thickness when compared to pigs treated with ultrasound and ODN-PESDA. Interobserver variability was 3% (n = 12 comparisons) for IVUS measurements and 14% (n = 25 comparisons) for repeated histological measurements.

[0134]

[Table 6]

[Brief description of the drawings]

FIG. 1 is a Lineweaver-Burk plot of binding data with PS-ODN for PESDA (sonicated dextrose / albumin exposed to perfluorocarbon) microbubbles. The equilibrium dissociation constant K _m for binding to microbubbles (calculated for seven concentrations performed in duplicate; see Example 5) is
It was 1.76 × 10 ⁻⁵ M. Sequence 5′d (AAC GTT GAG GGG
This value reported for the binding of the 15-mer PS-ODN with SEQ ID NO: 1 to human serum albumin in solution is almost 3.7-4.8.
X 10 ^-5 M (Srinivasan, SK et al., "Chara
cterization of Binding Sites, Extent
of Binding, and Drug Interactions of
Oligonucleotides with Album ", Antis
ense Res. Devel. 5: 131, 1995).

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] July 12, 2000 (2000.7.12)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction contents]

From the foregoing, it is understood that more effective targeted drug delivery systems for the delivery of biologics, especially polynucleotides and oligonucleotides, are required for these drugs to achieve their full potential. Can be done. Several references describe the use of gas-filled microbubbles as contrast agents
are doing. In US Patent No. 5,567,415, Porter
Ultrasound dextrose-albumin suspension while introducing fluorocarbon gas
Microbubbles prepared by processing are described. See Porter et al.
Am. Coll. Cardiol. 25 (2): 509, 1995) is air,
Nitrogen, helium or SF _6, (a) sonication in air followed by (b) sheet
Contains gas, exposed by continuous mixing with the selected gas in the syringe
Teaching microbubbles to be made. Other references describe microbubbles for delivery of therapeutic agents.
ing. Unger (WO 96/39197) describes in amphiphilic fluorinated compounds
It teaches drug delivery via encapsulated microbubbles. With encapsulated gas
Can include fluorocarbons, nitrogen, oxygen, or air. Typical
In the preparation, the microbubbles form a headspace over an aqueous suspension of the amphiphilic fluorinated compound.
Pour fluorocarbon and / or nitrogen in between and shake the container
Formed by Porter et al. (Circulation 96 (
8): L401, 1997) contains either air or fluorocarbon
Discloses microbubbles for delivery of oligonucleotides. Porter and I
versen (WO 98/00172) is a protein-encapsulated microparticle.
Compare the small bubbles, which are different gases: nitrogen, helium, and
Contains sulfur hexafluoride. This microbubble is sonicated in air, followed by hematological analysis.
Prepared by mixing with each gas in a locker. Marsden (WO
98/18498) for use in diagnostic imaging and / or therapy.
A targeted ultrasound contrast agent is described. This reference states that the drug
May include any of a number of gases, and are actually used in its 35 working examples.
Gas teaches that perfluorocarbon, SF _6, or air. P
orter et al. (WO 97/33474) disclose the use of ultrasound in combination with microbubbles.
It teaches the delivery of drugs by conjugation. PESDA (exposure to perfluorocarbon
Exposed and sonicated dextrose / albumin) microbubbles are
Prepared by manual stirring with carbon followed by sonication in air.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction contents]

SUMMARY OF THE INVENTION In one aspect, the invention provides a method for delivering a biological agent to a specific tissue site. The method comprises the steps of (a) forming an aqueous suspension of a plurality of microbubbles containing an insoluble gas, encapsulated with a protein, wherein the biological agent is in air. under conditions resulting in oxygen content, and N is lower than ₂ partial pressure N ₂ partial pressure of the small Awanai in this microbubble higher than the oxygen content of the micro Awanai obtained via sonication, Administering the suspension to an animal such that the protein directs the microbubble conjugating agent to a site of bioprocessing of the protein; And releasing the biological agent upon dissipation of the microbubbles. Preferably, the microbubbles are N ₂ depletion environment, and more preferably is formed in an environment containing no N ₂ (e.g., oxygen).

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction contents]

[0011] The present invention also provides related methods for delivering biological agents to specific tissue sites.
The method comprises the steps of (a)As described above,Insoluble gas encapsulated in protein
Forming an aqueous suspension of microbubbles containing water, wherein the biological suspension
The active agent is conjugated to the protein; (b) administering the suspension to the animal.
And (c) exposing the tissue site to ultrasound. this Microbubbles are contained within microbubbles obtained through sonication in air.From oxygen content
High oxygen content in this microbubble, andN_TwoN in this microbubble below the partial pressure_TwoMinute
It is formed under pressure-generating conditions. Therefore, the microbubbles are preferably N_TwoDepletion
Formed in the environment, and more preferably N_TwoEnvironment free from, for example, acids
Formed in the element.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventors Iversen, Patrick El. United States Nebraska 68127, Omaha, Wilson Drive 8226 (72) Inventor Mayer, Gary Dee. United States Ohio 45701, Asense, East Circle Drive 20, Suite 190 F-term (Reference) 4C076 AA22 AA64 AA95 BB12 CC11 CC41 DD35 EE41 EE59 FF68 4C084 AA11 AA13 MA05 MA23 MA38 NA05 NA06 NA13 ZA361 4C086 AA01 AA02 MA23 MA05 NA05 NA06 NA13 ZA36

Claims (22)

[Claims] 1. A method for delivering a biological agent to a specific tissue site, comprising: (a) forming an aqueous suspension of a plurality of microbubbles encapsulated with a protein and containing an insoluble gas. Wherein the biological agent has an oxygen content in the microbubbles higher than the oxygen content in the microbubbles obtained via sonication in air, and sonication in air. under conditions to produce N ₂ partial pressure in the lower fine vesiculation than N ₂ partial pressure of the micro Awanai obtained through, it is conjugated to the protein, process; and, (b) the suspension Administering a fluid to an animal such that the protein directs the drug to which the microbubble is conjugated to the bioprocessing site of the protein and releases the drug upon dissipation of the microbubble. A method comprising: 2. The method of claim 1, wherein the dissipation of the microbubbles is provided by exposing the tissue site to ultrasound. 3. A method for delivering a biological agent to a target site in an animal, comprising: (a) forming an aqueous suspension of a plurality of microbubbles encapsulated with a protein and containing an insoluble gas. Wherein the biological agent has an oxygen content in the microbubbles higher than the oxygen content in the microbubbles obtained via sonication in air, and sonication in air. under conditions to produce N ₂ partial pressure in the lower fine vesiculation than N ₂ partial pressure of the micro Awanai obtained through, it is conjugated to the protein, the step; and (b) the suspension Administering to the animal; and (c) applying an ultrasonic field to the target site. 4. The method of claim 1, wherein said microbubbles are formed in an N ₂ -free environment.
4. The method according to any one of the above items 3. 5. The method of claim 4, wherein said environment comprises oxygen. 6. The method according to claim 1, wherein the protein is selected from the group consisting of albumin, human gamma globulin, human apotransferrin, β-lactose and urease. 7. The method of claim 6, wherein said protein is albumin. 8. The method according to claim 1, wherein the insoluble gas is selected from the group consisting of perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, and perfluoropentane. 9. The biological agent according to claim 1, wherein the biological agent is a polynucleotide selected from the group consisting of an antisense oligonucleotide, an antigen oligonucleotide, an oligonucleotide probe, a vector, a virus vector, and a plasmid. The method according to any one of claims 1 to 4. 10. The microbubbles comprising: (a) using an aqueous dextrose solution containing 5% to 50% dextrose, using an aqueous solution of albumin containing about 2% to about 10% human serum albumin, about 2-fold step is diluted to about 8 times; (b) a step of agitating the solution with the insoluble gas; in and (c) N ₂ depleted environment, exposing the solution to sonication horn, solution in 4. A method according to any of the preceding claims, wherein the step of creating a cavity at the microparticle site, thereby producing stable microbubbles of about 0.1 to 10 microns in diameter. 11. The method of claim 10, wherein the N ₂ depleted environment in step (c) consists of oxygen. 12. The method of claim 11, wherein said oxygen is blown into an interface between said sonication horn and said solution. 13. A method for preventing carotid artery stenosis in an animal having a vascular injury, comprising: (a) aqueous suspension of a plurality of microbubbles encapsulated with a protein and containing an insoluble gas. Administering a suspension to the animal, wherein the protein is conjugated to an oligonucleotide that inhibits expression of a gene encoding a regulatory enzyme that mediates smooth muscle proliferation; and b) applying an ultrasonic field to the arterial site. 14. The microbubbles having an oxygen content in the microbubbles higher than the oxygen content in the microbubbles obtained through sonication in air, and through sonication in air. N ₂ partial pressure of the resulting micro Awanai formed under conditions which produce N ₂ partial pressure of a lower fine small in foam than the method of claim 13. 15. The method according to claim 13, wherein the gene is c-myc or c-myb. 16. A microbubble composition comprising an aqueous suspension of microbubbles encapsulated with a protein and containing an insoluble gas, wherein the microbubbles are obtained via sonication in air. oxygen content in the high fine small bubbles than the oxygen content in the microbubbles, and N ₂ partial pressure of a lower fine small in foam than N ₂ partial pressure of the micro Awanai obtained via sonication in air A microbubble composition comprising: 17. The composition of claim 16, further comprising a biological agent conjugated to said protein. 18. The composition according to claim 16, wherein said protein is selected from the group consisting of albumin, human gamma globulin, human apotransferrin, β-lactose and urease. 19. The composition according to claim 18, wherein said protein is albumin. 20. The method of claim 1, wherein the insoluble gas is a perfluorocarbon.
7. The composition according to 6. 21. The gas, wherein the gas is perfluoromethane, perfluoroethane,
21. The composition of claim 20, wherein the composition is selected from the group consisting of perfluoropropane, perfluorobutane, and perfluoropentane. 22. The method of claim 1, wherein the biological agent is an oligonucleotide.
8. The composition according to 7.